INTRODUÇÃO: O hipoestrogenismo pode determinar perda da massa mineral óssea, diminuindo a qualidade do osso. Assim, vários fármacos são ministrados para evitar esta perda, porém, podem determinar efeitos colaterais importantes. Portanto, questiona-se se o emprego do estrogênio associado a estas substâncias poderia minimizar os efeitos adversos e manteria a massa mineral óssea. Contudo, há poucas informações sobre os efeitos destas combinações. Esta pesquisa tem como objetivo avaliar a ação do estrogênio, raloxifeno e do extrato de soja rico em gensteína, isolado ou combinado no osso de ratas ovariectomizadas. MATERIAIS E MÉTODOS: No nono dia de nascimento, todas as ratas receberam propionato de testosterona (0,1 g/g). No sexto mês de idade, os animais do controle fisiológico foram identificados como GI e receberam apenas o veículo (propilenoglicol em 0,5 ml/dia) durante o experimento e os outros que receberam testosterona foram ovariectomizados e divididos aleatoriamente em seis grupos: GII veículo (controle castrado, n=6); GIII - estrogênio conjugados eqüinos (ECE, (50 g/Kg/dia, n=8); GIV raloxifeno (RAL, 0,75 mg/kg/dia, n=8); GV extrato de soja enriquecido com genisteína (ESG, 300 mg/kg/dia, n=7); GVI ECE + ESG (50 g/Kg/dia + 300 mg/kg/dia, n=7); GVII - ECE+RAL (50 g/Kg/dia + 0,75mg/kg/dia, n=6). Após três meses da cirurgia, os fármacos foram ministrados por 120 dias consecutivos. Posteriormente, os animais foram sacrificados sob anestesia, sendo retirada a tíbia esquerda para rotina histológica. Os cortes histológicos foram corados pela hematoxilina-eosina para avaliar a microarquitetura óssea. Foram feitos procedimentos imunoistoquímicos, de imunofluorescência e PCR para quantificar as principais proteínas ósseas estruturais (colágeno tipo I, osteocalcina, osteopontina e osteoprotegerina), bem como de seus respectivos RNA mensageiros. Os dados foram analisados pelos testes de ANOVA e Tukey. RESULTADOS: Todos os tratamentos determinaram aumento da quantidade de osso trabecular (p<0,05). As fibras totais de colágeno apresentaram-se aumentadas em todos os grupos tratados, exceto com o raloxifeno. Já as fibras finas de colágeno diminuíram apenas no grupo tratado com estrogênio. As frações de colágeno tipo I, mostraram-se aumentadas nos grupos tratados com estrogênio e sua asssociação com o raloxifeno. O colágeno tipo III esteve aumentado no grupo tratado com estrogênio em associação com extrato de soja rico em genisteína. Em relação às proteínas não colagenosas, a osteoprotegerina apresentou-se aumentada nos grupos tratados com estrogênio, suas associações e com o extrato de soja rico em genisteína. A osteopontina esteve diminuída em todos os grupos tratados e a osteocalcina mostrou-se aumentada apenas no grupo tratado com ralolxifeno, em comparação ao grupo castrado (p<0,05). Não houve diferença estatística significante do PCR em tempo real na análise dos transcritos entre os grupos estudados. CONCLUSÃO: A combinação de estrogênio com raloxifeno ou extrato de soja rico em genisteína não trouxe benefícios adicionais na qualidade do tecido ósseo, como ocorreu com esses fármacos isoladamente / INTRODUCTION: Hypoestrogenism can determine bone mineral loss, resulting in decreased bone quality. To prevent that process, several drugs are administered, which can lead, however, to important side effects. Therefore, it is questionable whether the use of estrogen associated with those substances could minimize the adverse effects and maintain bone mineral mass. There is little information on the effects of those compounds. This research aims to evaluate the action of unopposed estrogen or combined with raloxifene and genistein-rich soy extract on ovariectomized adult female rats. MATERIALS AND METHODS: On the ninth day of birth, rats received, testosterone propionate (0.1 mg / g). On the sixth month, animals in the physiological control were identified as GI and received only the vehicle (propylene glycol at 0.5 ml / day) during the experiment and the other which was administered testosterone underwent ovariectomy and divided randomly into six groups: GII - vehicle (control castrated, n = 6); GIII - conjugated equine estrogen (CEE, 50 mg / kg / day, n = 8); GIV - raloxifene (RAL, 0.75 mg / kg / day, n = 8) ; GV - soy extract enriched with genistein (ESG, 300 mg / kg / day, n = 7), GVI - ECE + ESG (50 mg / kg / day + 300 mg / kg / day, n = 7); GVII - ECE + RAL (50 mg / kg / day + 0.75mg/kg/day, n = 6).Three months after the surgery, drugs were consecutively administered for 120 days. Subsequently, the animals were sacrificed on anesthesia and their left tibiae were removed for routine histology. The histological sections were stained by hematoxylin-eosin to evaluate bone microarchitecture. Immunohistochemical, immunofluorescence and PCR procedures were performed to quantify the main structural bone proteins (type I collagen, osteocalcin, osteopontin, and osteoprotegerin) as well as their mRNA. The data were analyzed by ANOVA and Tukey test. RESULTS: All treatments led to increased amounts of trabecular bone (p <0.05). The total collagen fibers had to be enlarged in all treated groups, except with raloxifene. Already thin collagen fibers decreased only in the group treated with estrogen. The fractions of type I collagen, were increased in groups treated with estrogen and its asssociação with raloxifene. Type III collagen was increased in the group treated with estrogen in combination with soybean extract rich in genistein. Regarding the non-collagenous proteins, the increased osteoprotegerin presented in groups treated with estrogen, and their associations with soy extract rich in genistein. The osteopontin was decreased in all treated groups and osteocalcin was increased only in the treated group ralolxifeno, compared to the castrated group (p <0.05). There was no statistically significant difference from the real-time PCR analysis of transcribed between the groups. CONCLUSION: The combination of estrogen with raloxifene or genistein-rich soy extract was uncapable of bringing additional benefits to the quality of bone tissue as observed with those drugs alone

Le rachitisme vitamino-résistant hypophosphatémique (RVRH) est une maladie génétique rare causée par des mutations du gène PHEX. La perte de fonction de la protéine PHEX conduit à l’augmentation du FGF23, une hormone circulante qui agit sur le rein et entraîne une perte systémique de phosphate. Le squelette rachitique des patients atteints de RVRH présente des déformations osseuses et une ostéomalacie. La dentine hypominéralisée des patients est à l’origine d’abcès dentaires fréquents, mais le statut parodontal des patients RVRH est mal connu, de même que leur risque de développer une parodontite pouvant aboutir à la perte des dents. La fonction et le substrat de la protéine PHEX ne sont pas identifiés avec exactitude. Il a été montré in vitro que PHEX avait la capacité d’interagir et de dégrader des protéines membres de la famille des SIBLINGs comme MEPE ou OPN, toutes les deux impliquées dans la régulation de la minéralisation des tissus osseux et dentinaires, mais on ne sait pas si in vivo les défauts de minéralisation observés résultent principalement de l’hypophosphatémie systémique ou bien également des effets directs de l’absence de PHEX sur les protéines régulatrices de la minéralisation. L’objectif de cette thèse a consisté à s’intéresser à la physiopathologie du parodonte dans le RVRH ainsi qu’à déterminer quel était l’impact de la mutation de PHEX dans un modèle de biominéralisation humaine où les conditions de concentration en phosphate pouvaient être ajustées et normalisées. Nous avons d’abord analysé le statut parodontal de 34 patients RVRH dans une étude clinique cas-témoins et ainsi montré que les malades dont la supplémentation en phosphate et vitamine D était tardive ou incomplète présentaient une fréquence et une sévérité accrues de maladie parodontale. Le phénotype parodontal du RVRH a alors été étudié sur des échantillons humains et sur le modèle murin du RVRH, la souris HYP. Nous avons réalisé un modèle d’égression dentaire de façon à permettre une apposition du cément cellulaire, ainsi qu’un modèle de résorption et de réparation osseuses parodontales afin de caractériser l’impact du RVRH sur la physiopathologie parodontale. Nos résultats ont montré que le phénotype parodontal et sa physiopathologie étaient très perturbés dans le rachitisme vitamino-résistant hypophosphatémique et chez la souris HYP, nous avons aussi pu mettre en évidence que le rôle pathologique majeur joué par l’ostéopontine dans le tissu osseux au cours du RVRH ne pouvait pas être généralisé aux autres tissus minéralisés du parodonte. De façon à identifier le rôle de PHEX dans la minéralisation matricielle locale indépendamment de la phosphatémie systémique, nous avons ensemencé des matrices de collagène dense avec des cellules primaires humaines issues de patients RVRH comparés à des contrôles que nous avons cultivés pendant 24 jours en conditions ostéogéniques avec des concentrations en phosphate identiques. Nos résultats ont montré que malgré une concentration normale en phosphate, la perte de fonction de la protéine PHEX entraînait une diminution de la quantité et de la qualité de la phase minérale et une accumulation et une dégradation pathologiques de la protéine OPN. Les contributions originales de ce travail de thèse doctorale ont consisté à démontrer sur le plan clinique et biologique la susceptibilité accrue du rachitisme hypophosphatémique lié à l’X quant au risque de développer une maladie parodontale, ainsi qu’à apporter la preuve d’un rôle pathologique de l’absence de PHEX indépendant de la phosphatémie sur des cultures primaires humaines. / X-linked hypophosphatemia (XLH) is a rare X-linked dominant disorder caused by inactivating mutations in the PHEX gene. The impairment of PHEX protein leads to an increase in FGF23, a circulating factor that causes systemic loss of phosphate. The rachitic skeleton of patients with XLH displays short stature and osteomalacia. Dental defects include poorly mineralized dentin and spontaneous dental abscesses. Little is known about the periodontal condition of XLH and if patients are more prone to develop periodontitis, eventually leading to tooth loss. Although the exact function and substrate of PHEX are not known, it has been shown in vitro that PHEX could interact with SIBLING proteins such as MEPE or OPN, both involved in the regulation of bone and dentin mineralization, but it is not yet clear if the defects in the calcified extracellular matrices of XLH are caused by systemic hypophosphatemia only, or also by local consequences of the absence of PHEX. The aim of this doctoral dissertation was to explore the pathobiology of the XLH periodontium and to determine the impact of PHEX deficiency at the local level in a model of human biomineralization where phosphate supply could be adjusted and normalized. We first examined 34 adults with XLH in a case-control study and observed that periodontitis frequency and severity were increased in individuals with late or incomplete supplementation in phosphate and vitamin D analogs. The periodontium was then analyzed in XLH dental roots and further characterized in the Hyp mouse, the murine model of XLH. We performed a model of tooth movement adaptation leading to the formation of cellular cementum and a model of periodontal breakdown and repair to investigate the impact of XLH on the pathobiology of periodontal tissues. Our results showed strongly affected XLH/Hyp periodontal phenotype and impaired pathobiology and suggested that the key role played by OPN in bone could not be generalized to other periodontal mineralized tissues. In order to determine the role of PHEX in local human mineralization, dense collagen gels were seeded with primary human dental pulp cells harvested from XLH patients displaying PHEX mutations and age-matched healthy individuals. Cell-seeded gels were cultured up to 24 days under osteogenic conditions and controlled phosphate medium concentrations. Our results showed that despite normal phosphate concentrations, PHEX deficiency led to decreased quantity and quality of the mineral phase and a pathologic accumulation and processing of OPN. Overall the original contributions of this doctoral dissertation consist in the demonstration of a higher susceptibility of XLH patients to periodontitis and in the evidence of a local effect of PHEX deficiency in the pathologic intrinsic mineralization from XLH osteogenic cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A medula óssea adulta possui duas populações de células-tronco importantes no tratamento de diversas doenças hepáticas: células-tronco hematopoiéticas (CTHs) e células-tronco mesenquimais. A regeneração do fígado após a hepatectomia é um processo complexo que requer a proliferação de todas as células hepáticas. Fatores de crescimento, citocinas e componentes da matriz extracelular são elementos-chave nesse processo. As lamininas são uma família de proteínas de matriz extracelular, com funções adesivas e quimiotáticas pelo recrutamento de integrinas e outros receptores de superfície celular. No fígado normal, a laminina é expressa nas veias porta e centrolobular. O objetivo desse estudo foi investigar a expressão de laminina durante a regeneração hepática induzida por hepatectomia parcial e após o transplante de células mononucleares de medula óssea. As células mononucleares de medula óssea foram obtidas dos fêmures e tíbias de ratos, isoladas, marcadas com DAPI e injetadas pela veia porta em ratos recém-hepatectomizados. Os fígados foram coletados 15 minutos, 1 dia e 3 dias após a hepatectomia e o transplante de células de medula óssea e congelados. Os cortes foram imunomarcados com anticorpos primários anti-CD34 e anti-laminina de rato e observados em microscópio confocal de varredura a laser. Os resultados mostraram que 15 minutos após a hepatectomia parcial, as células-tronco hematopoiéticas CD34+ transplantadas foram encontradas em contato com a laminina localizada nas veias porta e centrolobular, indicando que a laminina poderia participar na adesão inicial das células-tronco a esses vasos logo após o seu transplante. Além disso, 1 e 3 dias após a hepatectomia, as células mononucleares de medula óssea transplantadas foram observadas nos sinusóides hepáticos expressando laminina. Esses resultados sugerem que a laminina pode ser um componente da matriz extracelular importante para a adesão e enxerto de células de medula óssea no fígado após uma lesão. Nós também analisamos a expressão de osteopontina (OPN) em células de medula óssea e CTHs. Os resultados por microscopia confocal demonstraram que a maioria das células mononucleares de medula óssea recém-isoladas expressa quantidades variáveis de OPN. Além disso, algumas CTHs CD34+ também expressam OPN. Após 1 e 4 dias de cultura, observamos uma diminuição de células expressando CD34, e um aumento na expressão de OPN pelas células mononucleares de medula óssea. / The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of heterotrimeric extracellular matrix proteins with adhesive and chemotactic functions, through recruitment of integrins and other cell surface receptors. In the normal liver, laminin is expressed in portal and centrolobular veins. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy and after bone marrow mononuclear cells transplantation. Rat bone marrow mononuclear cells were obtained from tibias and femurs, isolated, stained with DAPI and injected in immediately hepatectomyzed rats via portal vein. Livers were collected 15 minutes, 1 day and 3 days after hepatectomy and bone marrow cells transplantation and frozen. Liver sections were immunolabeled with mouse anti-rat CD34 and rabbit anti-rat laminin primary antibodies and observed under a Laser Scanning Confocal Microscope. Results showed that 15 minutes after partial hepatectomy, transplanted CD34+ HSCs were found in contact with laminin, which was localized principally in portal and centrolobular veins of rat livers, indicating that laminin could participate in stem cell initial attachment to these vessels soon after their transplantation. Furthermore, 1 and 3 days after hepatectomy, transplanted bone marrow mononuclear cells were found in the hepatic sinusoids expressing laminin. These results suggest that laminin could be an important extracellular matrix component for bone marrow cell adhesion and grafting in the injured liver. We also analyzed osteopontin (OPN) expression in bone marrow mononuclear cells and HSCs. Results by confocal microscopy showed that the most freshly isolated bone marrow mononuclear cells express variable amounts of OPN. Furthermore, some CD34+ HSCs also expressed OPN. After 1 and 4 days in culture, we observed a decrease in CD34+ cells, and an increase in OPN expression in bone marrow mononuclear cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A medula óssea adulta possui duas populações de células-tronco importantes no tratamento de diversas doenças hepáticas: células-tronco hematopoiéticas (CTHs) e células-tronco mesenquimais. A regeneração do fígado após a hepatectomia é um processo complexo que requer a proliferação de todas as células hepáticas. Fatores de crescimento, citocinas e componentes da matriz extracelular são elementos-chave nesse processo. As lamininas são uma família de proteínas de matriz extracelular, com funções adesivas e quimiotáticas pelo recrutamento de integrinas e outros receptores de superfície celular. No fígado normal, a laminina é expressa nas veias porta e centrolobular. O objetivo desse estudo foi investigar a expressão de laminina durante a regeneração hepática induzida por hepatectomia parcial e após o transplante de células mononucleares de medula óssea. As células mononucleares de medula óssea foram obtidas dos fêmures e tíbias de ratos, isoladas, marcadas com DAPI e injetadas pela veia porta em ratos recém-hepatectomizados. Os fígados foram coletados 15 minutos, 1 dia e 3 dias após a hepatectomia e o transplante de células de medula óssea e congelados. Os cortes foram imunomarcados com anticorpos primários anti-CD34 e anti-laminina de rato e observados em microscópio confocal de varredura a laser. Os resultados mostraram que 15 minutos após a hepatectomia parcial, as células-tronco hematopoiéticas CD34+ transplantadas foram encontradas em contato com a laminina localizada nas veias porta e centrolobular, indicando que a laminina poderia participar na adesão inicial das células-tronco a esses vasos logo após o seu transplante. Além disso, 1 e 3 dias após a hepatectomia, as células mononucleares de medula óssea transplantadas foram observadas nos sinusóides hepáticos expressando laminina. Esses resultados sugerem que a laminina pode ser um componente da matriz extracelular importante para a adesão e enxerto de células de medula óssea no fígado após uma lesão. Nós também analisamos a expressão de osteopontina (OPN) em células de medula óssea e CTHs. Os resultados por microscopia confocal demonstraram que a maioria das células mononucleares de medula óssea recém-isoladas expressa quantidades variáveis de OPN. Além disso, algumas CTHs CD34+ também expressam OPN. Após 1 e 4 dias de cultura, observamos uma diminuição de células expressando CD34, e um aumento na expressão de OPN pelas células mononucleares de medula óssea. / The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of heterotrimeric extracellular matrix proteins with adhesive and chemotactic functions, through recruitment of integrins and other cell surface receptors. In the normal liver, laminin is expressed in portal and centrolobular veins. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy and after bone marrow mononuclear cells transplantation. Rat bone marrow mononuclear cells were obtained from tibias and femurs, isolated, stained with DAPI and injected in immediately hepatectomyzed rats via portal vein. Livers were collected 15 minutes, 1 day and 3 days after hepatectomy and bone marrow cells transplantation and frozen. Liver sections were immunolabeled with mouse anti-rat CD34 and rabbit anti-rat laminin primary antibodies and observed under a Laser Scanning Confocal Microscope. Results showed that 15 minutes after partial hepatectomy, transplanted CD34+ HSCs were found in contact with laminin, which was localized principally in portal and centrolobular veins of rat livers, indicating that laminin could participate in stem cell initial attachment to these vessels soon after their transplantation. Furthermore, 1 and 3 days after hepatectomy, transplanted bone marrow mononuclear cells were found in the hepatic sinusoids expressing laminin. These results suggest that laminin could be an important extracellular matrix component for bone marrow cell adhesion and grafting in the injured liver. We also analyzed osteopontin (OPN) expression in bone marrow mononuclear cells and HSCs. Results by confocal microscopy showed that the most freshly isolated bone marrow mononuclear cells express variable amounts of OPN. Furthermore, some CD34+ HSCs also expressed OPN. After 1 and 4 days in culture, we observed a decrease in CD34+ cells, and an increase in OPN expression in bone marrow mononuclear cells.